Inward rectifier K+ channels (Kir channel proteins) are involved in the control of many physiological processes that are important to human health. Kir channel proteins normally function as K+ (potassium) selective pores that span cell membranes. The Kir channels are referred to as inward rectifier K+ (Kir) channels based on a fundamental ion conduction property of these channels: given an equal but opposite electrochemical driving force K+ conductance into the cell far exceeds conductance out of the cell.
Among their many functions Kir channel proteins control the pace of the heart, regulate secretion of hormones into the blood stream, generate electrical impulses underlying information transfer in the nervous system and control airway and vascular smooth muscle tone. It is believed that various disease states are directly related to the function of Kir channel proteins. Members of this channel family include Kir1-Kir7, (Kubo et al., Pharmacological Rev., 57:509-526, 2005) Hypertension, atrial fibrillation, and type 2 diabetes are related to Kir channel protein function and are serious conditions for which new therapies are needed. Specific links between Kir channel proteins and disease have been found. Kir1.1 channels are present in the kidney and regulate salt secretion into the urine. Heritable mutations involving Kir1.1 cause Barter's syndrome and hypotension. Compounds which selectively inhibit Kir1.1 have the potential to serve as a new form of anti-hypertensive agent in which hypokalemia, a major side-effect of currently used diuretics, should in principle not be a problem. Thus, hypertensive individuals could benefit from Kir1.1 inhibitor-based therapies. Kir3.1 and Kir3.4 channels, which assemble to form a heteromultimer, are called G-protein-gated K+ channels (GIRK). These channels control heart rate through stimulation by the parasympathetic nervous system. GIRK channel knock-out mice do not develop atrial fibrillation under any of the usual stimuli that induce this arrhythmia in mice. (Claphan et al., JACC 37, 2136-2143 (Jun. 15, 2001)) Accordingly, inhibition of GIRK channels in humans might provide effective treatment for atrial fibrillation. Kir6 channels are expressed in beta cells of the pancreas and control insulin secretion. With the identification of compounds that selectively inhibit the Kir6 channel new therapies could be realized for the treatment of type 2 diabetes. Accordingly, Kir channel proteins are good targets for the treatment of various diseases.
The Kir channel family of proteins are very similar to each other in both sequence and, by inference, structure; thus, it has been very difficult to identify compounds that can specifically modulate one kind of Kir channel protein without cross-reacting with other types of Kir channel proteins.
For the first time the structure of a eukaryotic Kir channel has been determined, and a structural feature “the turret region” has been identified that is highly ordered in structure and, based on the amino acid sequences will differ among Kir channels. Prior to this structure, only the structure of a prokaryotic Kir channel had been determined. (Nishida et al., EMBO, vol. 26, pp. 4005-4015 (2007)) The turret is an important functional region of the protein and faces the outside of the cell making this region an attractive target for identifying potential therapeutic compounds. Given the identification of the turret region in the various Kir channel proteins and the structure in a prototype, the present invention provides a variety of methods by which the turret region may be used to identify compounds having therapeutic utility for treating the various diseases related to the function of Kir channels.
The present invention provides for the first time the expression and purification of a eukaryotic Kir channel as explained in detail below. Study of the structure of this eukaryotic Kir channel resulted in a realization of the importance of the turret region and the invention of methods which allow identification of therapeutic compounds that can selectively bind to different members of the Kir channel family of proteins.